The presently disclosed inventive concepts relate generally to biological and environmental specimen sampling of macromolecules of interest and capture into a receiving device, e.g., a container or plate, for subsequent analysis, more particularly to laser-ablation-based sampling methods and devices, and specifically, to the sampling and analysis of macromolecules, such as nucleic acids, proteins and small peptides.
Rapid identification of harmful microorganisms (including human and animal pathogens) is of paramount importance to public health, national security and environmental safety. Most of the currently used methods rely on DNA recovery and polymerase chain reaction (PCR) amplification of selected DNA regions. The yield and quality of recovered DNA, as well as the presence of any PCR inhibitors, is of critical importance for the success of this approach. They vary considerably with DNA extraction procedures. Thus, the success of PCR-based methods for microbial identification is critically dependent on the DNA extraction strategy selected. Good-quality DNA is required to ensure clean PCR product sequences. Thus, a successful extraction method maintains the quality of DNA throughout the recovery process and minimizes the co-extraction of any compounds that inhibit the PCR. Currently used DNA recovery procedures can be time-consuming and often require a large quantity of starting material.
DNA isolation and purification protocols customized for different types of clinical (blood, tissue, saliva, urine, surface swabs) and environmental (soil, water, waste, sediments) specimens are available and often provided in a pre-packaged kit form. A step common to all DNA recovery methods is the disruption of cellular structures, followed by separation and purification of the DNA from the lysed material. Cell lysis can be achieved through a multitude of single or combined chemical and mechanical means, including enzymatic digestion, bead beating, sonication, application of detergents and solvents, as well as freezing and thawing1. Subsequent DNA purification steps may include centrifugation, filtration, selective adsorption on silica, or paramagnetic-particle technology2. With the latter system, DNA can be extracted from up to 16 samples in less than 45 minutes. Of major concern are method- and cell type-dependent differences in the efficacy of DNA recovery and, subsequently, its yield. For example when treated with an alkaline polyethyleneglycol, DNAzol® Direct3, which is also marketed as an universal agent for direct PCR (Molecular Research Center Inc., Cincinnati, Ohio), Gram-negative bacteria are lysed after approximately 15 minutes, while more resistant Gram-positive organisms may require up to 3 hours of incubation at room temperature to achieve cell disruption. This may result in variations in DNA recovery within samples that can result in incorrect information on microbial community structure, particularly in specimens harboring highly diverse populations, such as biofilms4. Furthermore, a large variety of compounds inhibit PCR5. The specimen itself can be a source of PCR-inhibiting compounds6, and it is essential that such compounds are removed during DNA sampling and extraction. However, it should be noted that successful PCR is sometimes possible without cleanup of lysates7.
Undoubtedly, strategies that would rapidly, i.e. within seconds, generate DNA that is ready for PCR amplification are of considerable interest to the life science community8. Owing to their unprecedented speed of collecting material from well-defined surface areas, laser ablation-based methods carry great promise for enabling such technology. Applying laser pulses to rapidly deposit a large amount of energy into biological materials and other organic substrates has been explored in a range of important applications across many scientific disciplines. For example, nanosecond, visible or ultraviolet, laser pulses have been employed to disrupt cells9,10. Such “optical lysis” has been found to be particularly useful for rupturing single cells11. Pulsed laser irradiation is a key step in matrix-assisted laser desorption ionization (MALDI) of biomolecules allowing their analysis with mass spectrometry12,13.